Powdery Mildew Resistance Providing Genes in Cucumis Sativus

ABSTRACT

The present invention relates to powdery mildew resistance providing genes of the  Cucumis  family, and especially  Cucumis sativus , wherein said resistance is provided by impairment of the present genes. Further, the present invention relates plants comprising the present impaired resistance conferring genes and seeds, embryos or other propagation material thereof. Especially, the present invention relates to powdery mildew resistance conferring genes, wherein the amino acid sequence encoded by said resistance conferring gene is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 and SEQ ID No. 22, and amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of co-pending U.S. patentapplication Ser. No. 14/002,284, filed Oct. 29, 2013, which is the U.S.national phase of PCT Application No. PCT/EP2012/052483, filed Feb. 20,2012, which claims priority to PCT Application No. PCT/EP2011/053054,filed Mar. 1, 2011, each of which is incorporated herein by reference intheir entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 1604498_ST25.txt. The size of thetext file is 109,661 bytes, and the text file was created on Sep. 16,2016.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to powdery mildew resistance providinggenes of Cucumis sativus, wherein said resistance is provided byimpairment of the present genes either at the expression or proteinlevel. Further, the present invention relates to plants comprising thepresent resistance conferring genes and seeds, embryos or otherpropagation material thereof.

Powdery mildew (PM) is one of the main fungal diseases known in plantsbelonging to the Cucumis family such as Cucumis sativus(cucumber), bothin the field and greenhouse.

Powdery mildew diseases are generally caused by many different speciesof fungi of the order Erysiphales. The disease is characterized bydistinctive symptoms such as white powder-like spots on the leaves andstems. Generally, the lower leaves are the most affected, but the mildewcan appear on any part of the plant that is exposed above ground. As thedisease progresses, the spots get larger and thicker as massive numbersof spores form, and the mildew spreads up and down the length of theplant such as on the stem and even the fruits.

Severely affected leaves can become dry and brittle, or can wither anddie. Because of the infection, the fruits can be smaller in size, fewerin number, less able to be successfully stored, sun scalded,incompletely ripe, and having a poor flavor. It may also predisposeplants to be more vulnerable to other pathogens. Eventually, the plantcan die.

Powdery mildew can, amongst others, be caused by the fungus Sphaerothecafuliginea (recently renamed: Podosphaera xanthii also designated asOidium erysiphoides) and/or Erysiphe cichoracearum DC (recently renamed:Golovinomyces cichoracearum also designated as Oidium chrysanthemi).

Considering the economic importance of Cucumis plant species, such ascucumber, there is a continued need in the art to provide powdery mildewresistance providing genes.

In view of the above need in the art, it is an object of the presentinvention, amongst other objects, to meet this need.

SUMMARY OF THE INVENTION

According to the present invention, this object, amongst other objects,is met by a powdery mildew resistance conferring gene as defined in theappended claim 1.

Specifically, this object of the present invention, amongst otherobjects, is met by a powdery mildew resistance conferring gene, whereinthe amino acid sequence encoded by said resistance conferring gene isselected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ IDNo. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 18, SEQ ID No. 20 and SEQ ID No. 22, and amino acidsequences with more than 70% identity, preferably more than 80%identity, more preferably more than 90% identity, and most preferablymore than 95% identity such as more than 96%, 97%, 98%, 99%; and whereinsaid resistance conferring gene is impaired.

The object of the present invention, amongst other objects, isadditionally met by a powdery mildew resistance conferring gene, whereinthe cDNA sequence transcribed from said resistance conferring gene isselected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ IDNo. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21, and cDNAsequences with more than 70% identity, preferably more than 80%identity, more preferably more than 90% identity, and most preferablymore than 95% identity such as more than 96%, 97%, 98%, 99%; and whereinsaid resistance conferring gene is impaired.

Impaired resistance conferring gene according to the present inventionis meant to indicate a gene providing a reduced, or even absent,susceptibility to powdery mildew caused by fungi indicated bypowder-like spots on the leaves and stems, such as fungi belonging tothe order Erysiphales such as Sphaerotheca fuliginea (recently renamed:Podosphaera xanthii also designated as Oidium erysiphoides) and/orErysiphe cichoracearum DC.

Impaired resistance conferring genes according to the present inventionare mutated genes. The mutation of the present genes can, throughdifferent mechanisms, result in impairment. For example, mutations inprotein encoding DNA sequences may lead to mutated, truncated ornon-functional proteins. Mutations in non-coding DNA sequences may causealternative splicing, translation or protein trafficking. Alternatively,a mutation resulting in an altered transcriptional activity of a gene,which determines the amount of mRNA available for translation toprotein, may results in low levels, or absence, of proteins.Additionally, the impairment of gene function may be caused aftertranslation, i.e. at protein level.

Impairment according to the present invention is also indicated byobserving a powdery mildew resistance in a Cucumis sativus plantcomprising a gene which as mutated at the protein level as compared tothe SEQ ID Nos. provided herein or no expression of the SEQ ID Nos.provided herein is observed.

Impaired is also indicated herein as a non-functional gene or protein.Although the function of the present genes is not yet identified, anon-functional gene or protein can be readily determined by establishingpowdery mildew resistance (non-functional) or powdery mildewsusceptibility (functional) in a plant. A powdery mildew resistance(non-functional) plant is indicated by comprising a gene which asmutated at the protein level as compared to the SEQ ID Nos. providedherein or no expression of the SEQ ID Nos. provided herein is observed.

Functional and non-functional genes or proteins can also be determinedusing complementation experiments. For example, transforming a resistantpowdery mildew Cucumis sativus plant with any of the present genes orproteins will result in a powdery mildew susceptible Cucumis sativusplant when the gene or protein is functional while the Cucumis sativusplant will remain resistant when the gene or protein is non-functional.

According to the present invention, the present powdery mildewresistance conferring genes provide powdery mildew resistance when thepresent genes are impaired. Impaired according to the present inventioncan be indicated by the absence, or decrease of a functional, ornon-muted, protein identified herein as SEQ ID No. 2, SEQ ID No. 4, SEQID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQID No. 16, SEQ ID No. 18, SEQ ID No. 20 or SEQ ID No. 22. In the art,many mechanisms are known resulting in the impairment of a gene eitherat the transcription, translation or protein level.

For example, impairment at the transcription level can be the result ofone or more mutations in transcription regulation sequences, such aspromoters, enhancers, and initiation, termination or intron splicingsequences. These sequences are generally located 5′ of, 3′ of, or withinthe coding sequence represented by SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ IDNo. 15, SEQ ID No. 17, SEQ ID No. 19, or SEQ ID No. 21. Impairment canalso be provided by a deletion, rearrangement or insertion in thepresent genes.

Impairment at the translation level can be provided by a prematurestop-codons or other RNA->protein controlling mechanisms (such assplicing) or posttranslational modifications influencing, for example,protein folding or cellular trafficking.

Impairment at the protein level can be provided by truncated, misfoldedor disturbed protein-protein interactions.

Independent of the underlying mechanism, impairment according to thepresent invention is indicated by a decrease or absence a functionalprotein according to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ IDNo. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQID No. 18, SEQ ID No. 20 or SEQ ID No. 22.

According to a preferred embodiment, impairment according to the presentinvention is provided by one or more mutations in the present genesresulting in the absence of a protein expression product. As indicated,these mutations can cause a defective expression at the transcription ortranslation level.

According to another preferred embodiment, impairment according to thepresent invention is caused by one or more mutations in the presentgenes resulting in a non-functional protein expression product. Anon-functional protein expression product can, for example, be caused bypremature stop-codons, incorrect translation or posttranslationalprocessing or by insertions, deletions or amino acid changes.

Using molecular biology methods, impairment of the present genes canalso be accomplished by gene silencing, for example using siRNA orknocking out of the present genes. Methods based on EMS or othermutagenic chemical compounds capable of randomly changing nucleic acidsinto other nucleotides are also contemplated within the context of thepresent invention. Detection of such mutations typically involves highsensitivity melting curve analyses or nucleotide sequencing-basedTILLING procedures.

The present invention relates to nucleotide and amino acid sequenceswith more than 70%, preferably more than 80%, more preferably more than90% and most preferably more than 95% sequence identity either at thenucleotide level or the amino acid level.

Sequence identity as used herein is defined as the number of identicalconsecutive aligned nucleotides, or amino acids, over the full length ofthe present sequences divided by the number of nucleotides, or aminoacids, of the full length of the present sequences and multiplied by100%.

For example, a sequence with 80% identity to SEQ ID No. 1 comprises overthe total length of 1782 nucleotides of SEQ ID No. 15 1426 identicalaligned consecutive nucleotides, i.e., 1426/1782*100%=80%.

According to the invention, the present genes are derived from Cucumissativus.

According to another aspect, the present invention relates to Cucumissativus plants comprising in their genome the present impaired powderymildew resistance conferring genes, i.e., plants not expressing afunctional protein selected from the group consisting of SEQ ID No. 2,SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12,SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20 and SEQ IDNo. 22, and amino acid sequences with more than 70% identity, preferablymore than 80% identity, more preferably more than 90% identity, and mostpreferably more than 95% identity.

In general, and preferably, the present plants will be homozygous forthe present impaired genes, i.e., comprising two impaired powdery mildewresistance conferring genes, wherein the cDNA sequence transcribed fromsaid resistance conferring gene is selected from the group consisting ofSEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9,SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No.19, and SEQ ID No. 21, and cDNA sequences with more than 70% identity,preferably more than 80% identity, more preferably more than 90%identity, and most preferably more than 95% identity.

Considering the benefits of the present plants, i.e., providing powderymildew resistance in cucumber plants, the invention also relates toseeds, plant parts or propagation material capable of providing thepresent powdery mildew resistant cucumber plants which seeds, plantparts or propagation material comprise one or more of the presentpowdery mildew resistance conferring genes, i.e., impaired powderymildew resistance conferring genes, wherein the cDNA sequencetranscribed from said resistance conferring gene is selected from thegroup consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No.7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No.17, SEQ ID No. 19, and SEQ ID No. 21, and cDNA sequences with more than70% identity, preferably more than 80% identity, more preferably morethan 90% identity, and most preferably more than 95% identity.

According to yet another aspect, the present invention relates toisolated nucleotide sequences selected from the group consisting of SEQID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ IDNo. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, andSEQ ID No. 21, and nucleotide sequences with more than 70% identity,preferably more than 80% identity, more preferably more than 90%identity, and most preferably more than 95% identity.

According to still another aspect, the present invention relates toisolated amino acid sequences selected from the group consisting of SEQID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20and SEQ ID No. 22, and amino acid sequences with more than 70% identity,preferably more than 80% identity, more preferably more than 90%identity, and most preferably more than 95% identity.

The present invention also relates to the use of one or more of thepresent powdery mildew resistance conferring genes, one or more of thepresent isolated nucleotide sequences, or one or more of the presentisolated amino acid sequences for providing a powdery mildew resistantcucumber plant (Cucumis sativus). As indicated, the present use is basedon impairment, either at the expression or protein level, of the genesdescribed herein and can be readily determined by the presently providedcDNA and amino acid sequences optionally in combination withdetermination of the presence or absence of powdery mildew resistanceand/or in combination with complementation assays.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described in the examples below ofpreferred embodiments of the present invention. In the example,reference is made to figures wherein:

FIG. 1: shows Relative expression levels of CsKIP2 in a selection ofcucumber germplasm. Average values of expression per group either inabsence (−) or presence (+) of a transposon are added including standarddeviation error bars. Bar colors indicate the reference gene used tomake the calculations.

FIG. 2: shows DNA fragments of CsKIP2 amplified with primers ID3 (SEQ IDNO: 25) and ID4 (SEQ ID NO: 26) and visualized by gel electrophoresis(2% agarose, 10 v/cm, 40 minutes)

FIG. 3: shows cDNA sequence alignment of lines with absence (top strand,SEQ ID NO: 27) and presence (bottom strand, SEQ ID NO: 28) of atransposon-like element in CsKIP2 genomic DNA. Here, a 72 bp deletion isvisible in cDNA derived from lines with the transposon-like elementpresent. Primer binding positions are shown in bold italic characters

FIG. 4: shows an amino acid alignment of CsKIP9 alleles of powderymildew susceptible and powdery mildew resistant CsKIP9 alleles. Theamino acid sequences shown in FIG. 4 correspond with the following SEQID Nos.:

CsMlo9OK561 SEQ ID No: 29 CsMlo9OK537 SEQ ID No: 30 CsMlo9OK619 SEQ IDNo: 31 CsMlo9OK123 SEQ ID No: 32 CsMlo9OK563 SEQ ID No: 33 Consensus SEQID No: 34

DESCRIPTION OF THE INVENTION Examples Example 1 Cucumber GermplasmScreen for Contribution of CsKIP2 Expression Levels and Allelic Variantsto Resistance/Susceptibility Introduction

Impairment of functioning of genes can be caused by differentmechanisms. Mutations in protein encoding DNA sequences may be causalfor loss-of-function alleles or genes with a change in characteristics.Alternatively, altered transcriptional activity of a gene, whichdetermines the amount of mRNA available for translation to protein, mayresult in low levels of available proteins. Additionally, the impairmentof gene function may be caused after translation, i.e. at protein level.

The present example shows that a mutation (deletion) in the codingsequence of CsKIP2 provides powdery mildew resistance.

Material and Methods

A total of twelve Cucumber germplasm lines varying in powdery mildewresistance levels were selected for analysis. Seeds were germinatedunder standard greenhouse conditions. Hypocotyls were infected with alocal powdery mildew isolate 7 days past sowing followed by infection ofthe first true leaf 14 days past sowing. Evaluation of reactionphenotypes was performed 28 days past sowing (21 and 14 days postinfection for hypocotyls) and phenotypes are scored on a scale of 1-9,where 1 is fully susceptible and 9 is fully resistant.

Material of infected plants was collected for subsequent RNA isolationaccording to standard procedures (Machery-Nagel RNA Plant). RNAisolation was followed by cDNA synthesis using a standard Oligo-dTprimer combined with a reverse transcriptase (Finnzymes) with 1 μg totalRNA input.

Expression levels of CsKIP2 were determined with CsKIP2 specific PCRprimer pair (table 1, ID1 ID2) amplifying a DNA fragment from exon 5 toexon 7 with a size specific to cDNA, highly different of the productsize derived from genomic DNA. In addition, control fragmentsfunctioning as internal reference were amplified from three differenthousekeeping genes i.e. Elongation Factor 1-alpha (EF-1, A. thalianaortholog At1g07920.1), Protein Phosphatase 2a subunit a2 (PDF2, A.thaliana ortholog At3g25800.1) and Helicase domain containing protein 1(HEL1, A. thaliana ortholog At1g58050.1).

For specific real time detection of dsDNA during PCR amplification,LCGreen (Idaho Technologies) was added to the PCR reaction mixture at0.5× concentration. Calculations were made using the ΔΔCt method.

For the detection of allelic variants with CsKIP2, a specific region wastargeted in the cDNA. This region is suspected to house atransposon-like element (exon 11 transposon). Primers (table 1, ID3ID4)) designed to specifically amplify exon 9 (partial), 10 and 11(partial) of CsKIP2 were used for the detection of this fragment.

TABLE 1 CsKIP2 specific PCR primers ID1: 5′CGACACTTGAGCTTCTGGAG 3′SEQ ID NO. 23 ID2: 5′GCAAGATGTGCAACAATGAATC 3′ SEQ ID NO. 24 ID3:5′CCCGCAATGTGGCTATTTGCTGT 3′ SEQ ID NO. 25 ID4:5′CCCGAGGCTGAACGACCGGA 3′ SEQ ID NO. 26

Results

A selection of 12 germplasm lines from the powdery mildew disease testwas made for subsequent expression studies and detection of allelicvariation of CsKIP2. Presence or absence of a transposon-like elementsuspected to be the causative factor of powdery mildew resistance ingenomic DNA was done before starting expression studies in order toinvestigate its effect on expression.

Expression of CsKIP2 in leaf material derived from the selected plantswas determined based on the control genes. The results show no generaleffect of expression based on the obtained data. On average, expressionlevels observed are similar (FIG. 1)

After determination of expression levels, the allelic variation in thetransposon-like element was investigated. The fragment amplified withprimers ID3 and ID4 produced a variable fragment of size 199 bp or 127bp (FIG. 2). The smaller fragment was found strictly in resistant plantsand correlates to presence of the transposon in genomic DNA.

The sequence of the fragments was found to be highly similar except fora 72 bp deletion in exon 11, centered around the original position ofthe transposon in genomic DNA (FIG. 3).

CONCLUSIONS

Expression analysis of CsKIP2 in leaf material from infected cucumberplants was carried out in order to assess the involvement of geneexpression in resistance. On average, expression levels were similar inresistant and susceptible plants.

The presence of a transposon-like element found in exon 11 of the CsKIP2gene in genomic DNA was also found not to be correlated to expressionlevels of CsKIP2.

The presence of the transposon-like element in genomic DNA was found tobe related to resistance of plants to powdery mildew. The resistantplants with the transposon-like element in the genomic DNA showed adeletion of 72 bp in exon 11 in the cDNA, compared to susceptibleplants.

Apparently, the mechanism responsible for the correct splicing of RNA(i.e. separating exons from introns), splices the transposon-likeelement from the RNA, along with a part (i.e. 72 bp) of the codingsequence. After translation of mRNA to protein, the 72 bp deletion mRNAresults in a protein with a 24 amino acid residue deletion. The 24 aminoacid residue deletion protein product (i.e. CsKIP2 from resistantplants) is believed to have lost its function as host-factor.

Example 2 Cucumber Germplasm Screen for Contribution of CsKIP9 AllelicVariants to Resistance/Susceptibility

The cDNA sequence of CsKIP9 of 5 cucumber plants was determined. Table 2below summarizes the plants tested and their powdery mildew resistance.

TABLE 2 Cucumber plants used for cDNA sequencing of CsKIP9 Cucumberplant Powdery mildew resistant SEQ ID No. OK561 − 29 OK537 + 30 OK619 +31 OK123 + 32 OK563 − 33

The amino acid sequences encoded by the cDNAs were aligned as shown inFIG. 4. An amino acid substitution of asparagine (N) by aspartic acid(D) (LEEN to LEED) at position 284 of CsKIP9 (SEQ ID No. 2) was found tocorrelate with the powdery mildew resistance observed.

Example 3 Powdery Mildew Resistant Cucumber Plant with Non-FunctionCsKIP9

The cDNA sequence CsKIP9 of a powdery mildew resistant cucumber plantwas determined and an amino acid substation in exon 3 was defined.Specifically, the coding sequence of the first amino acids of exon 3(positions 61 to 63 of SEQ ID No. 2) of functional CsKIP9 are Glutamicacid (E)-Leucine (L)-Methionine (M) [ELM]. However, in the powderymildew resistant cucumber plant identified, this sequence was mutated toAlanine (A)-Threonine (T)-Isoleucine (I) [ATI] indicating that thissubstitution in CsKIP9 correlates with the powdery mildew resistanceobserved.

The powdery mildew resistance providing genes identified herein aresummarized in table 3 below. The cDNA and amino acid sequences providedare the powdery mildew resistant genes in their functional form, i.e.providing powdery mildew resistance when impaired at the protein level,such as by mutation, or impaired at the expression level.

TABLE 3 cDNA and Amino Acid Sequences of the Present Genes Gene IdentityPlant Sequence type SEQ ID No. CsKIP9 Cucumis sativus cDNA 1 Aa 2 CsKIP2Cucumis sativus cDNA 3 aa 4 CsKIP1 Cucumis sativus cDNA 5 aa 6 CsKIP3Cucumis sativus cDNA 7 aa 8 CsKIP4 Cucumis sativus cDNA 9 aa 10 CsKIP5Cucumis sativus cDNA 11 aa 12 CsKIP6 Cucumis sativus cDNA 13 aa 14CsKIP7 Cucumis sativus cDNA 15 aa 16 CsKIP8 Cucumis sativus cDNA 17 aa18 CsKIP10 Cucumis sativus cDNA 19 aa 20 CsKIP11 Cucumis sativus cDNA 21aa 22

What is claimed is:
 1. An isolated cucumber plant that is resistant topowdery mildew comprising in its genome an impaired powdery-mildewsusceptibility gene, wherein the presence of the impaired powdery-mildewsusceptibility gene is determinable by an absence or loss of function ofSEQ ID NO: 4 compared to a cucumber plant that is susceptible to powderymildew, wherein the impairment is one or more non-natural mutations thatcause at least one of an absence or loss of function of SEQ ID NO:
 4. 2.The isolated cucumber plant according to claim 1, wherein the impairmentcauses lengthening of an N-terminal region of SEQ ID NO: 4, wherein thelengthening comprises amino acids 2-16 of SEQ ID NO: 31(LFYNANFVQKSRLFQ).
 3. The isolated cucumber plant according to claim 1,wherein the impairment is a deletion in a coding region of a nucleicacid sequence having the sequence SEQ ID NO:
 3. 4. The isolated cucumberplant according to claim 3, wherein the deletion is in exon
 11. 5. Theisolated cucumber plant according to claim 3, wherein the deletion is ofat least 72 base pairs.
 6. The isolated cucumber plant according toclaim 1, wherein the non-natural mutation causes a shortening of SEQ IDNO:
 4. 7. The isolated cucumber plant according to claim 6, wherein SEQID NO: 4 is shortened by 24 amino acids.
 8. A seed, fruit, plant part,or propagation material of the cucumber plant according to claim 1,wherein the seed, fruit, plant part, or propagation material comprisesthe one or more non-natural mutations of SEQ ID NO: 3 and exhibitsresistance to powdery mildew.
 9. A method for obtaining a cucumber plantthat is resistant to powdery mildew, comprising introducing animpairment comprising one or more non-natural mutations to a nucleotidesequence having a sequence of SEQ ID NO: 3 in a cucumber plant that issusceptible to powdery mildew, wherein the impairment causes at leastone of an absence or loss of function of SEQ ID NO:
 4. 10. The methodaccording to claim 9, wherein the one or more non-natural mutationscause the absence of SEQ ID NO:
 4. 11. The method according to claim 9,wherein the and wherein the one or more non-natural mutations cause anon-functioning SEQ ID NO:
 4. 12. A plant produced by the methodaccording to claim 9, wherein the plant comprises the one or morenon-natural mutations of SEQ ID NO: 3 and exhibits resistance to powderymildew.
 13. A seed, fruit, plant part, or propagation material of thecucumber plant produced according to the method of claim 9, wherein theseed, fruit, plant part, or propagation material comprises the one ormore non-natural mutations of SEQ ID NO: 3, and wherein the seed, fruit,plant part, or propagation material exhibits resistance to powderymildew.